Growth promotion method

ABSTRACT

The present invention relates to a method of promoting growth comprising the step of administering an effective amount of a compound of formula I:  
                 
in which X and Y are either the same or different and selected from a heteroatom; 
            is a double or single bond depending on the heteroatoms X and Y;    R 1  to R 5  are either the same or different and selected from hydrogen or a non-deleterious substituent; and    R 6  and R 7  are either the same or different and selected from hydrogen and a non-deleterious substituent or one of R 6  and R 7  are absent when there is a double bond present, pharmaceutically or veterinarily acceptable salts or derivatives, pro-drugs, tautomers and/or isomers thereof to a subject in need thereof.

FIELD OF THE INVENTION

This invention relates to a growth promotion method and in particular toa method of promoting growth of an animal, more specifically intensivelyfarmed animals, such as, pigs, cows, sheep, chickens, turkeys or fish.

BACKGROUND

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

A popular method of livestock management is the intensive managementsystem, which dramatically increases the population density of farmedanimals and therefore improves the cost efficiency of housing,management and labour compared to less intensive management systems.However, a major consequence of intensive management is the increasedopportunity for the development and spread of disease. This disease maybe caused by specific pathogens introduced, for example, by errors inpractice management, or by opportunistic pathogens in the contaminatedand compromising intensively managed environment.

Clinical infections can lead to major disease outbreaks with highmorbidity and/or mortality. Subclinical infections can interfere withthe normal metabolic process of an animal. For example, interferencewith the gastrointestinal function of an animal can interfere with itsnormal eating habits. This results in poor growth and feed efficienciesby the animal, which in turn results in less efficient animalproduction.

Most additives can, but do not always, improve feed use and rate of liveweight gain by 3 to 8 percent. However, as the additives increase thecost of a diet, their inclusion will only be cost effective if theimproved performance covers the cost of the additive and the sameimprovement could not have been obtained by a less expensive means, suchas a change in management system or an improved strain of animal.

Current feed additives approved for use in livestock diets are generallyadministered at 1 to 175 mg/kg feed. Many feed additives must bewithdrawn from the diet before slaughter in order to minimise thepresence of residues of the additive in the meat of the animal whenconsumed.

Current additives used to promote growth of animals include theadministration of antibiotics, vitamins and minerals including copperand arsenicals, and hormone regulation therapies. Classic examplesinclude use of the arsenicals to control swine dysentery, coccidiostatsto control coccidiosis in poultry, and vitamins which compensate forvitamin deficiencies. However, feeding these molecules to animals whichwill be consumed by humans often raises concerns on the effect to bothhumans and the environment as a result of the development of antibioticresistant bacteria and the residues remaining in the animal and in thesoil. For example, there has been evidence of copper residues in piglivers and also in soils treated with pig slurry.

The development of antibiotic resistance in organisms is another majorconcern to consumers and compromises the ability of many widely usedantibiotics to treat serious infections in humans. Not all antibioticspromote growth. For example, broad spectrum antibiotics are notgenerally used for this purpose. Therefore, the development of a growthpromoting agent which leaves a minimal or no detectable residue is alsoof concern to consumers. Allergic reactions which occur as a result ofconsuming specific additives is known to occur. The effect of hormonesupplements on consumers is of further concern and several hormonetreatment programs in poultry and beef cattle have been restricted.There is increasing pressure from consumers to restrict the use of manycompounds. In the European Economic Community, for example,non-authorised additives include tetracyclines, penicillins andcephalosporins, aminoglycosides, macrolides, sulfonamides andtrimethoprim, nitrofurans except nitrovin, arsenicals, hormones andanti-hormones.

A requirement accordingly exists for growth promoting agents which leaveminimal residues in animals to which they have been administered and inthe environment.

SUMMARY OF INVENTION

We have now found that certain substituted nitrostyrene compounds haveimproved growth promotant activity, require low dosage, result in lowtissue levels, and have low oral toxicity.

In a first aspect, the present invention provides a method of promotinggrowth comprising the step of administering an effective amount of acompound of

in which

X and Y are either the same or different and selected from a heteroatom;

is a double or single bond depending on the heteroatoms X and Y;

R₁ to R₅ are either the same or different and selected from hydrogen ora non-deleterious substituent; and

R₆ and R₇ are either the same or different and selected from hydrogenand a non-deleterious substituent or one of R₆ and R₇ are absent whenthere is a double bond present,

pharmaceutically or veterinarily acceptable salts or derivatives,pro-drugs, tautomers and/or isomers thereof to a subject in needthereof.

The present invention also provides the use of the compound of formula Iin promoting growth of a subject.

The present invention further provides the use of the compound offormula I in the manufacture of a medicament or feed for promotinggrowth of a subject.

The present invention still further provides the compound of formula Ifor use in promoting growth of a subject.

In a second aspect, the present invention provides a composition forpromoting growth in a subject, which comprises the compound of formula Iand a carrier.

When the composition is to be administered to a human or animal, it ispreferably in the form of a pharmaceutical or veterinary compositioncomprising the compound of formula I and a pharmaceutically orveterinarily acceptable carrier.

Alternatively, the composition may be administered in the form of a feedfor promoting growth in a subject comprising the compound of formula I.

In a third aspect, the present invention provides a growth promotingagent or a nutritional supplement comprising the compound of formula I.

The present invention also provides use of the compound of formula I asa growth promoting agent or nutritional supplement.

DETAILED DESCRIPTION OF THE INVENTION

In the subject specification, except where the context requiresotherwise due to express language or necessary implication, the words“comprise” or variations such as “comprises” or “comprising” are used inan inclusive sense, i.e. to specify the presence of the stated featuresbut not to preclude the presence or addition of further features invarious embodiments of the invention.

It must be noted that, as used in the subject specification, thesingular forms “a”, “an” and “the” include plural aspects unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a single compound, as well as two or more compounds;and so forth.

In the compound of formula I, preferably X and Y are either the same ordifferent and selected from O and N, more preferably both X and Y areoxygen.

Preferably R₁ and R₂ are either the same or different and selected fromhydrogen, hydroxy, halogen and optionally substituted C₁₋₆ alkyl.

R₃ to R₅ are preferably either the same or different and selected fromhydrogen, hydroxy, halogen, nitro, C₁₋₆ alkoxy and optionallysubstituted C₁₋₆ alkyl.

Preferably halogen is chlorine or bromine.

The E isomer of the compounds of formula I is preferred.

Particularly preferred are compounds of the formula I in which X, Y,

, R₆ and R₇ are as defined above; R₁ and R₂ are either the same ordifferent and selected from hydrogen, hydroxy, Cl, Br and C₁₋₄ alkyl;and R₃ to R₅ are either the same or different and selected fromhydrogen, hydroxy, Cl, Br, nitro, C₁₋₄ alkoxy and C₁₋₄ alkyl.

Specific examples of the compounds of the present invention are asfollows:

-   -   (1) X and Y are O, R₁ is methyl and R₂ to R₇ are hydrogen        (3,4-methylenedioxy-β-methyl-β-nitrostyrene) (hereinafter        referred to as “Iksin”)    -   (2) X and Y are O and R₁ to R₇ are hydrogen        (3,4-methylenedioxy-β-nitrostyrene)    -   (3) X is N, Y is NH, R₁ is methyl, R₂ to R₆ are hydrogen and R₇        is absent (benzimidazole-5-β-nitropropylene)    -   (4) X is N, Y is NH, R₁ to R₅ are hydrogen, R₆ is methyl and R₇        is absent (2-methyl benzimidazole-5-β-nitroethylene)    -   (5) X is O, Y is N, R₁ to R₆ are hydrogen and R₇ is absent        (benzoxazole-5-β-nitroethylene)    -   (6) X is N, Y is O, R₁ is methyl, R₂ to R₅ are hydrogen, R₆ is        methyl and R₇ is absent (2-methyl        benzoxazole-5-β-nitropropylene)

By “pharmaceutically acceptable derivative” is meant anypharmaceutically acceptable salt, hydrate, ester, amide, activemetabolite, analogue, residue or any other compound which is notbiologically or otherwise undesirable and induces the desiredpharmacological and/or physiological effect.

The salts of the compound of formula I are preferably pharmaceuticallyacceptable, but it will be appreciated that non-pharmaceuticallyacceptable salts also fall within the scope of the present invention,since these are useful as intermediates in the preparation ofpharmaceutically acceptable salts. Examples of pharmaceuticallyacceptable salts include salts of pharmaceutically acceptable cationssuch as sodium, potassium, lithium, calcium, magnesium, ammonium andalkylammonium; acid addition salts of pharmaceutically acceptableinorganic acids such as hydrochloric, orthophosphoric, sulphuric,phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; orsalts of pharmaceutically acceptable organic acids such as acetic,propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric,lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic,methanesulphonic, trihalomethanesulphonic, toluenesulphonic,benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic,stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic andvaleric acids.

In addition, some of the compounds of the present invention may formsolvates with water or common organic solvents. Such solvates areencompassed within the scope of the invention.

The term “pro-drug” is used herein in its broadest sense to includefunctional derivatives of the compound of formula I which are readilyconvertible in vivo to the compound of formula I. Conventionalprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in “Design of Prodrugs” ed. H.Bundgaard, Elsevier, 1985.

The term “tautomer” is used herein in its broadest sense to includecompounds of formula I which are capable of existing in a state ofequilibrium between two isomeric forms. Such compounds may differ in thebond connecting two atoms or groups and the position of these atoms orgroups in the compound.

The term “isomer” is used herein in its broadest sense and includesstructural, geometric and stereo isomers. As the compound of formula Imay have one or more chiral centres, it is capable of existing inenantiomeric forms.

The term “heteroatom” denotes O, N or S.

The term “non-deleterious substituent” is used herein in its broadestsense and refers to a substituent which does not have a deleteriouseffect on the growth promoting property of the compound. Examplesinclude alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl,haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy,haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl,nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino,alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino,diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl,arylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy,arylsulphenyloxy, heterocyclyl, heterocycloxy, heterocyclamino,haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy,carboaryloxy mercapto, alkylthio, arylthio, acylthio andphosphorus-containing compounds.

Particularly suitable non-deleterious substituents are alkyl, alkenyl,alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, hydroxy, alkoxy,alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, nitroalkyl, nitroalkenyland nitroalkynyl.

In a preferred embodiment the non-deleterious substituents are C₁₋₆alkyl, halo, hydroxy, C₁₋₆ alkoxy and nitro.

The term “halogen” refers to fluorine, chlorine, bromine and iodine,preferably chlorine and bromine.

The term “alkoxy” is used herein in its broadest sense and refers tostraight chain, branched chain or cyclic oxy-containing radicals eachhaving alkyl portions, preferably C₁₋₆ alkyl, more preferably C₁₋₄alkyl. Examples of such alkoxy groups are methoxy, ethoxy, propoxy,butoxy and t-butoxy.

The terms “C₁₋₄ alkyl” or “C₁₋₆ alkyl” used either alone or in compoundwords such as “optionally substituted C₁₋₄ or C₁₋₆ alkyl” refer tostraight chain, branched chain or cyclic hydrocarbon groups having from1 to 6 carbon atoms. Illustrative of such alkyl groups are methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, neopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl.

The term “subject” as used herein refers to any subject in which thepromotion of growth is desired. The subject may have or be at risk ofhaving a disease or condition which requires growth promotion. Thesubject may be an animal or a human. While it is particularlycontemplated that the compounds of the invention are suitable fortreatment of domestic animals such as horses, ponies, donkeys, mules,llama, alpaca, pigs, cattle, sheep, birds and fish, or zoo animals suchas primates, felids, canids, bovids, and ungulates, they are alsoapplicable to use in humans and companion animals such as dogs and cats.

Preferably the animal is a mammal, bird or fish.

Suitable mammals include members of the orders Primates, Rodentia,Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Membersof the orders Perissodactyla and Artiodactyla are particularly preferredbecause of their economic importance.

For example, Artiodactyla comprises approximately 150 living speciesdistributed through nine families: pigs (Suidae), peccaries(Tayassuidae), hippopotamuses (Hippopotamidae), camels (Camelidae),chevrotains (Tragulidae), giraffes and okapi (Giraffidae), deer(Cervidae), pronghorn (Antilocapridae), and cattle, sheep, goats andantelope (Bovidae). Many of these animals, such as goats, sheep, cattleand pigs have very similar biology and share high degrees of genomichomology.

The order Perissodactyla comprises horses and donkeys, which are botheconomically important and closely related. Indeed, it is well knownthat horses and donkeys interbreed.

Preferably the mammal is a pig, cow or sheep.

Examples of birds include members of the order Anseriformes, whichincludes geese and ducks; Galliformes, which includes chickens, turkeys,quail, pheasants, guinea fowl and pea fowl; and Columbiformes, whichincludes pigeons and doves. Preferably the bird is a chicken, turkey,duck or goose.

Examples of fish include Clupeiforms, Perciformes, Gadiformes,Pleuronectiformes, Cypriniformes, Crustaceans and Molluscs.

In a preferred embodiment, the subject is an animal, more preferably anintensively farmed animal such as mammals, for example, pigs, cows orsheep; birds, for example, chickens such as broiler chickens, turkeys,ducks or geese; or fish.

As used herein, the term “farmed intensively” means farmed with the aimof achieving maximum production within a limited area.

Without wishing to be bound be theory, it is believed that compounds offormula I act to promote the growth of a subject by inhibiting thegrowth of pathogens in the gastrointestinal tract as well as having adirect effect of feed utilisation.

The promotion of growth may be determined by any suitable known method.In a preferred method, promotion of growth is determined by an increasein the weight, length, and/or height or a decrease in food intake, areduction in time to reach marketable weight and/or instance ofmicrobial infection of the subject as compared to a control subject. Ina more preferred method, promotion of growth is determined by anincrease in the weight of an subject as compared to a control subject.

It will be clearly understood that the terms “promotion of growth” and“promoting growth” mean the increase of growth of an subject whencompared to a control subject.

As used herein, the term “control subject” means a subject of the samespecies, age and sex as the subject whose growth is being promotedhowever, the control subject has not been administered the compound offormula

As used herein, the term “effective amount” is meant an amount of acompound of the present invention effective to yield the desired growthpromoting activity.

The specific “effective amount” will, obviously, vary with such factorsas the particular condition being treated, the physical condition of thesubject, the type of subject being treated, the duration of thetreatment, the nature of concurrent therapy (if any), the specificformulations employed and the structure of the compound or itsderivatives.

Dosage levels of the compound of formula I of the present invention maybe of the order of up to about 1 g per kilogram body weight. The amountof active ingredient that may be combined with the carrier materials toproduce a single dosage will vary depending upon the animal treated andthe particular mode of administration. For example, a formulationintended for oral administration to humans may contain up to about 1 gof an active compound with an appropriate and convenient amount ofcarrier material which may vary from about 5 to about 95 percent of thetotal composition. Dosage unit forms will generally contain between fromabout 5 mg to about 500 mg of active ingredient. When used in a feed,the amount of active ingredient will be about 10 ppm to about 100 ppm.

Optionally the compounds of the invention are administered in a divideddose schedule, such that there are at least two administrations in totalin the schedule. Administrations are given preferably at least every twohours for up to four hours or longer; for example the compound may beadministered every hour or every half hour. In one preferred embodiment,the divided-dose regimen comprises a second administration of thecompound of the invention after an interval from the firstadministration sufficiently long that the level of active compound inthe blood has decreased to approximately from 5-30% of the maximumplasma level reached after the first administration, so as to maintainan effective content of active agent in the blood. Optionally one ormore subsequent administrations may be given at a corresponding intervalfrom each preceding administration, preferably when the plasma level hasdecreased to approximately from 10-50% of the immediately-precedingmaximum.

The compounds of the present invention may additionally be combined withother molecules to provide an operative combination. It is intended toinclude any chemically compatible combination of pharmaceutically- orveterinarily-active agents, as long as the combination does noteliminate the activity of the compound of formula I. It will beappreciated that the compound of the invention and the other molecule(s)may be administered separately, sequentially or simultaneously.

Other molecules which may be used to promote growth include otherantimicrobials in reduced amounts, vitamins and/or minerals.

As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering thecompound of formula I to a subject. The carrier may be liquid or solidand is selected with the planned manner of administration in mind. Eachcarrier must be pharmaceutically or veterinarily “acceptable” in thesense of being compatible with other ingredients of the composition andnon-injurious to a subject.

The pharmaceutically or veterinarily acceptable carrier is preferably anorganic solvent such as acetone, benzene, acetonitrile, DMSO or analcohol, for example, methanol or ethanol. While the compounds offormula I show a poor solubility in water, when water is combined withan organic solvent a stable mixture is formed.

Carriers suitable for use in the preparation of the feed supplementsinclude alfalfa meal, soybean meal, cottonseed oil meal, linseed oilmeal, sodium chloride, corn meal, cane molasses, urea, bone meal, fishmeal, corncob meal, calcium chloride, vegetable or plant oils such asolive oil or canola oil and other similar materials. Use of the carriersin feed supplements promote uniformity of distribution of the compoundof formula I in the finished feed into which the supplement is blended.It thus performs an important function by ensuring proper distributionof the compound of formula I throughout the feed.

It will be understood that the compound of formula I can be administeredby any means. Preferably, the compound is administered orally,topically, or parentally, more preferably orally, most preferably orallyin the form of a feed. The compound of formula I may be administeredorally, topically, or parenterally in dosage unit formulationscontaining conventional non-toxic pharmaceutically or veterinarilyacceptable carriers, adjuvants, and vehicles. The term parenteral asused herein includes subcutaneous injections, aerosol for administrationto lungs or nasal cavity, intravenous, intramuscular, intrathecal,intracranial, injection or infusion techniques.

The present invention also provides suitable topical, oral andparenteral pharmaceutical or veterinary formulations for use in themethods of the present invention. The compounds of the present inventionmay be administered orally as a feed, or as tablets, aqueous or oilysuspensions, lozenges, troches, powders, granules, emulsions, capsules,syrups or elixirs. As used herein, the word “feed” means food or drink.

The composition for oral use may contain one or more agents selectedfrom the group of sweetening agents, flavouring agents, colouring agentsand preserving agents in order to produce pharmaceutically orveterinarily elegant and palatable preparations. Suitable sweetenersinclude sucrose, lactose, glucose, aspartame or saccharin. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.Suitable flavouring agents include peppermint oil, oil of wintergreen,cherry, orange or raspberry flavouring. Suitable preservatives includesodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methylparaben, propyl paraben or sodium bisulphite. Suitable lubricantsinclude magnesium stearate, stearic acid, sodium oleate, sodium chlorideor talc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate. The tablets contain the active ingredient inadmixture with non-toxic pharmaceutically or veterinarily acceptableexcipients which are suitable for the manufacture of tablets.

These excipients may be, for example, (1) inert diluents, such ascalcium carbonate, lactose, calcium phosphate or sodium phosphate; (2)granulating and disintegrating agents, such as corn starch or alginicacid; (3) binding agents, such as starch, gelatin or acacia; and (4)lubricating agents, such as magnesium stearate, stearic acid or talc.These tablets may be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. Coating may also be performed using techniques described inthe U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

The compound of formula I as well as the pharmaceutically orveterinarily active agent useful in the method of the invention can beadministered, for in vivo application, parenterally by injection or bygradual perfusion over time independently or together. Administrationmay be intravenously, intraarterial, intraperitoneally, intramuscularly,subcutaneously, intracavity, transdermally or infusion by, for example,osmotic pump. For in vitro studies the agents may be added or dissolvedin an appropriate biologically acceptable solvent or buffer and added toa cell or tissue.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable or plant oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, other antimicrobials, anti-oxidants, chelating agents, growthfactors and inert gases and the like.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a disease or sign orsymptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure of a disease. “Treating” as used herein covers anytreatment of, or prevention of disease in a subject, and includes: (a)preventing the disease from occurring in a subject that may bepredisposed to the disease, but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; or (c)relieving or ameliorating the effects of the disease, i.e., causeregression of the effects of the disease.

The invention includes various pharmaceutical or veterinary compositionsuseful for ameliorating disease. The pharmaceutical or veterinarycompositions according to one embodiment of the invention are preparedby bringing the compound of formula I, analogues, derivatives or saltsthereof, or combinations of the compound of formula I and one or morepharmaceutically or veterinarily active agents into a form suitable foradministration to a subject using carriers, excipients and additives orauxiliaries. Frequently used carriers or auxiliaries include magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk protein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial agents, anti-oxidants, chelating agents and inert gases.Other pharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 20th ed. Williams & Williams (2000), the British NationalFormulary, 43^(rd) edition (British Medical Association and RoyalPharmaceutical Society of Great Britain, 2000), the contents of whichare hereby incorporated by reference. The pH and exact concentration ofthe various components of the pharmaceutical or veterinary compositionare adjusted according to routine skills in the art. See Goodman andGilman's The Pharmacological Basis for Therapeutics (7th ed., 1985).

The pharmaceutical or veterinary compositions are preferably preparedand administered in dose units. Solid dose units may be tablets,capsules and suppositories. For treatment of a subject, depending onactivity of the compound, manner of administration, nature and severityof the disorder, age and body weight of the animal, different dailydoses can be used. Under certain circumstances, however, higher or lowerdaily doses may be appropriate. The administration of the daily dose canbe carried out both by single administration in the form of anindividual dose unit or else several smaller dose units and also bymultiple administration of subdivided doses at specific intervals.

The pharmaceutical or veterinary compositions according to the inventionmay be administered locally or systemically in a therapeuticallyeffective dose. Amounts effective for this use will, of course, dependon the severity of the disease and the weight and general state of thesubject. Typically, dosages used in vitro may provide useful guidance inthe amounts useful for in situ administration of the pharmaceutical orveterinary composition, and animal models may be used to determineeffective dosages for the promotion of growth. Various considerationsare described, e.g., in Langer, Science, 249: 1527, (1990). Formulationsfor oral use may be in the form of hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin. They may also be in theform of soft gelatin capsules wherein the active ingredient is mixedwith water or an oil medium, such as peanut oil, liquid paraffin orolive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspension. Suchexcipients may be (1) suspending agent such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2)dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The pharmaceutical or veterinary compositions may be in the form of asterile injectable aqueous or oleagenous suspension. This suspension maybe formulated according to known methods using those suitable dispersingor wetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono-or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compounds of formula I may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

The compounds of formula I may preferably be presented for use in theform of veterinary compositions, which may be prepared, for example, bymethods that are conventional in the art. Examples of such veterinarycompositions include those adapted for: oral administration, externalapplication, for example drenches (e.g. aqueous or non-aqueous solutionsor suspensions); tablets or boluses; powders, granules or pellets foradmixture with feed stuffs; pastes for application to the tongue;parenteral administration for example by subcutaneous, intramuscular orintravenous injection, e.g. as a sterile solution or suspension; or(when appropriate) by intramammary injection where a suspension orsolution is introduced in the udder via the teat; topical applications,e.g. as a cream, ointment or spray applied to the skin; orintravaginally, e.g. as a pessary, cream or foam.

BRIEF DESCRIPTION OF THE DRAWINGS

In the examples, reference will be made to the accompanying drawings inwhich:

FIG. 1 is a graph showing plasma levels of Iksin in chicks aftertreatment with Iksin at 100 μg/g in DMSO and at 400 μg/g in distilledwater/Tween20; and

FIG. 2 is a graph showing the average percentage weight increase inchicks after single dosing with Iksin at 12 μg/g in DMSO, 100 μg/g inDMSO, 200 μg/g in distilled water/Tween20 and 400 μg/g in distilledwater/tween20 compared to untreated chicks.

EXAMPLES

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

Example 1 Determination of Dose Range

Day-old chicks were orally administered Iksin in DMSO at 100 μg/g, 125μg/g, 150 μg/g and 200 μg/g body weight and in Distilled Water (DW)/5%Tween-20 at 100 μg/g, 200 μg/g, 400 μg/g and 500 μg/g body weight.

The maximum tolerated oral dose of Iksin for day-old broiler chicks was200 μg/g Iksin/DMSO and ≧500 μg/g in DW/T20.

Very little Iksin was absorbed from a single oral dosing. Iksin bloodlevels for doses of 12 μg/g DMSO or 200 μg/g DW were below the limits ofdetection. At 100 μg/g DMSO and 400 μg/g DW peak levels of 850 μg/g wereobserved at 48 h (FIG. 1).

Effect on Chick Growth

Treatment with all doses and formulations of Iksin resulted in increasedweight gain in chicks 4 days after a single treatment (FIG. 2). Chickstreated with Iksin 100 μg/g DMSO showed a 32% increase in weight gainover the group of control chicks. Treatment with Iksin 12 μg/g DMSO, 200and 400 μg/g DW/T20 gave a mean weight gain of 17%.

Conclusions

Oral administration of Iksin, formulated both in water and DMSO, iseffective in promoting chick growth and is poorly absorbed with lowtissue residues. It is anticipated that the results observed in thechicks will also be observed in other animals and humans.

Example 2 Repeat Poultry Trial

Objective

To investigate the potential of Iksin as a performance enhancing feedadditive for broiler chickens and to determine some safety criteria forits use.

Purpose of Trial

The purpose of this trial is to compare the performance of Iksinsupplemented feed against negative control feed (no additive) andpositive control feed (Zn bacitracin).

The parameters to be measured are

(i) weight gain and feed conversion rates at weekly intervals;

(ii) pathogen colonisation in the gastrointestinal tract with Salmonellaand Campylobacter by cloacal swab during rearing and by caecum contentsat time of processing;

(iii) the residue of Iksin in breast muscle tissue, fat and liver attime of processing and after 5 days withdrawal; and

(iv) the level of residues in faeces and broiler litter at time ofprocessing and after 5 days withdrawal.

Trial Protocol and Methods

Manufacture of Rations

Standard no-additive rations of Broiler Starter and Grower-FinisherFeeds (Ridley) supplemented with Iksin in canola oil and Zn bacitracin(commercial Albac 150) at the concentration recommended by themanufacturer will be prepared by Longerenong Agricultural College.

Chickens

720 1-day old commercial broiler chicks from Hazeldene Bendigo.

Treatment Groups

Three treatment groups (240 birds each) with 4 replicates (60 birdseach)

-   1 rations supplemented with Iksin at 50 mg/kg-   2 rations supplemented with Zn bacitracin-   3 rations with no additives-   OR-   Six treatment groups of 2 replicates each (or 3 replicates +1)

1a Iksin 50 mg/kg+old litter

-   1b Iksin 50+new litter-   2a Zn bacitracin+old litter-   2b Zn bacitracin+new litter-   3a No additives+old litter-   3b No additives+new litter    Broiler Chick Management

Birds will be housed according to the industry standard for commercialbroiler flocks spatial requirements (0.67 sq ft/bird) and environmentalconditions.

Each replicate will be separately housed with separate feed and watersupplies.

Feed and water will be available ad libitum.

Pens will be seeded with broiler litter (16 weeks old) from a commercialrearing shed at Longerenong.

This litter will be assayed for presence of Clostridium perfringens,Salmonella enterica and Campylobacter spp to confirm contaminationlevel.

Broiler litter will be handled according to commercial industrypractice.

Iksin treated birds and litter will be destroyed to waste according toenvironmental standards. Untreated birds and positive control birds willbe processed as broilers for human consumption.

Birds for Serial Monitoring

Five chicks from each replicate in each treatment group will be randomlyselected for the collection of specific data throughout the study.

Specific Data

a) measurement of individual weight gain at 0, 14, 28 and 42 days (orslaughter);

b) Collection of cloacal swabs at 0, 14, 28, 42 days (or slaughter) formicrobiological culture for the presence of Campylobacter andSalmonella;

c) Collection of breast muscle, liver and fat tissue at slaughter fromIksin treated birds+samples of broiler litter for measurement of Iksinresidues;

d) Collection of breast muscle, liver and fat tissue+samples of broilerlitter at 5 days post-withdrawal for measurement of Iksin residues;

e) Collection of faecal samples from Iksin treated birds for assay forIksin;

f) Collection of caecal contents and caecal tissue to determinecolonisation and invasion respectively with Campylobacter andSalimonella (mean viable count per treatment group).

General Data

a) Record of feed consumed between 0, 14, 28, 42 days (or slaughter) byweighback of feed in hoppers.

b) Observation for appearance, behaviour and general health andwell-being.

c) Record of morbidity (deaths+unfit culls) and any gross pathologydata.

Anlysis of Data

Weight gain and feed efficiency between treatment groups will becompared.

Pathogen colonisation rate in sentinel birds from all treatment groupswill be determined by presence/absence of pathogens in cloacal samples.

The effect of Iksin treatment on Campylobacter colonisation (caecalcontents) and invasion (caecal tissue) at slaughter will be compared tocontrols of the treatment group mean viable counts.

Expected Outcomes

The trial should show:

(i) whether there is a weight gain advantage over negative control birdsat specific intervals and at time of processing;

(ii) how Iksin compares to a treatment regimen previously used in theindustry;

(iii) whether Iksin reduces pathogen incidence (Salmonella andCampylobacter) in the gastrointestinal tract during rearing;

(iv) whether Iksin reduces pathogen loads or invasion by Campylobacterand salmonella into poultry tissues;

(v) whether there are any Iksin residues in muscle, fat and livertissues at slaughter and after withdrawal;

(vi) the level of Iksin in broiler litter at slaughter and afterwithdrawal; and

(vii) whether there is any effect on growth rates of the use of cleanversus contaminated litter.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

After entry into the U.S. national stage, and assurance of a U.S. filingdate, the present document revises the claims from the PCT applicationby amending claims 1, 2, 5, 6, 8-16, 23, 24 and 25, and canceling claims17-22. According to 37 C.F.R. § 1.121(c), after entry of the presentamendment, the status of the claims in the case is as follows:

1. A method of promoting growth in a subject in need thereof, comprisingadministering a compound of formula I:

in which X and Y are either the same or different and selected from aheteroatom;

is a double or single bond depending on the heteroatoms X and Y; R₁ toR₅ are either the same or different and selected from hydrogen or anon-deleterious substituent; and R₆ and R₇ are either the same ordifferent and selected from hydrogen and a non-deleterious substituentor one of R₆ and R₇ are absent when there is a double bond present, orpharmaceutically or veterinarily acceptable salts or derivatives,pro-drugs, tautomers and/or isomers thereof, to said subject in anamount effective to promote growth in said subject.
 2. A methodaccording to claim 1, in which the subject is an animal.
 3. A methodaccording to claim 2, in which the animal is an intensively farmedanimal.
 4. A method according to claim 3, in which the intensivelyfarmed animal is a mammal, bird or fish.
 5. A method according to claim4, in which the mammal is a pig, cow or sheep.
 6. A method according toclaim 1, in which X and Y are either the same or different and selectedfrom O and N.
 7. A method according to claim 6, in which X and Y areboth O.
 8. A method according to claim 1, in which R₁ and R₂ are eitherthe same or different and selected from hydrogen, hydroxy, halogen andoptionally substituted C₁₋₆ alkyl.
 9. A method according to claim 1, inwhich R₃ to R₅ are either the same or different and selected fromhydrogen, hydroxy, halogen, nitro, C₁₋₆ alkoxy and optionallysubstituted C₁₋₆ alkyl.
 10. A method according to claim 8 in which thehalogen is chlorine or bromine.
 11. A method according to claim 1, inwhich the compound of the formula I is in the form of an E isomer.
 12. Amethod according to claim 11, in which X, Y,

, R₆ and R₇ are as defined in claim 1; R₁ and R₂ are either the same ordifferent and selected from hydrogen, hydroxy, Cl, Br and C₁₋₄ alkyl;and R₃ to R₅ are either the same or different and selected fromhydrogen, hydroxy, Cl, Br, nitro, C₁₋₄ alkoxy and C₁₋₄ alkyl.
 13. Amethod according to claim 1, in which X and Y are O, R₁ is methyl and R₂to R₇ are hydrogen (3,4-methylenedioxy-β-methyl-β-nitrostyrene)

X and Y are O and R₁ to R₇ are hydrogen(3,4-methylenedioxy-β-nitrostyrene)

X is N, Y is NH, R₁ is methyl, R₂ to R₆ are hydrogen and R₇ is absent(benzimidazole-5-β-nitropropylene)

X is N, Y is NH, R₁ to R₅ are hydrogen, R₆ is methyl and R₇ is absent(2-methyl benzimidazole-5-β-nitroethylene)

X is O, Y is N, R₁ to R₆ are hydrogen and R₇ is absent(benzoxazole-5-β-nitroethylene)

X is N, Y is O, R₁ is methyl, R₂ to R₅ are hydrogen, R₆ is methyl and R₇is absent (2-methyl benzoxazole-5-β-nitropropylene)


14. A method according to claim 1, in which the subject is a human. 15.A method according to claim 4, in which the bird is a chicken or turkey.16. A method according to claim 9, in which the halogen is chlorine orbromine. 17.-22. (canceled)
 23. A feed comprising the compound offormula I as defined in claim 1 and a carrier selected from alfalfameal, soybean meal, cottonseed oil meal, linseed oil meal, corn meal,cane molasses, urea, bone meal, fish meal and corncob meal.
 24. A methodof promoting growth in a bird, comprising administering to said bird anamount of a compound effective to promote growth in said bird, whereinsaid compound is a compound of formula I:

in which X and Y are either the same or different and selected from aheteroatom;

is a double or single bond depending on the heteroatoms X and Y; R₁ toR₅ are either the same or different and selected from hydrogen or anon-deleterious substituent; and R₆ and R₇ are either the same ordifferent and selected from hydrogen and a non-deleterious substituentor one of R₆ and R₇ are absent when there is a double bond present, or apharmaceutically or veterinarily acceptable salt thereof.
 25. Use of thecompound of formula I as defined in any one of claims 1 to 13 as agrowth promoting agent or nutritional supplement A method according toclaim 24, in which the bird is a chicken or turkey.